bims-minimp 2022-02-27 papers

on Mitochondria, innate immunity, proteostasis

Issue of 2022‒02‒27
fourteen papers selected by
Hanna Salmonowicz
International Institute of Molecular Mechanisms and Machines of the Polish Academy of Sciences

The Pet127 protein is a mitochondrial 5'-to-3' exoribonuclease from the PD-(D/E)XK superfamily involved in RNA maturation and intron degradation in yeasts.
Brown adipose TRX2 deficiency activates mtDNA-NLRP3 to impair thermogenesis and protect against diet-induced insulin resistance.
Aberrant cyclin C nuclear release induces mitochondrial fragmentation and dysfunction in MED13L syndrome fibroblasts.
Analysis of Mitochondrial Function, Structure, and Intracellular Organization In Situ in Cardiomyocytes and Skeletal Muscles.
Bcl-2 Family Members and the Mitochondrial Import Machineries: The Roads to Death.
C19orf12 ablation causes ferroptosis in mitochondrial membrane protein-associated with neurodegeneration.
Redox-dependent loss of flavin by mitochondria complex I is different in brain and heart.
Mitochondrial and metabolic remodeling in human skin fibroblasts in response to glucose availability.
Disruption of Mitochondrial Complex I Induces Progressive Parkinsonism.
Stress-responsive regulation of extracellular proteostasis.
ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation.
Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation.
Mitochondrial COA7 is a heme-binding protein with disulfide reductase activity, which acts in the early stages of complex IV assembly.
Mitochondrial dysfunction associated with TANGO2 deficiency.

RNA. 2022 Feb 23. pii: rna.079083.121. [Epub ahead of print]

The Pet127 protein is a mitochondrial 5'-to-3' exoribonuclease from the PD-(D/E)XK superfamily involved in RNA maturation and intron degradation in yeasts.

Karolina Labedzka-Dmoch, Michal Razew, Marta Gapinska, Jakub Piatkowski, Adam Kolondra, Hanna Salmonowicz, Joanna M Wenda, Marcin Nowotny, Pawel Golik.

  Pet127 is a mitochondrial protein found in multiple eukaryotic lineages, but absent from several taxa, including plants and animals. Distant homology suggests that it belongs to the divergent PD-(D/E)XK superfamily which includes various nucleases and related proteins. Earlier yeast genetics experiments suggest that it plays a nonessential role in RNA degradation and 5' end processing. Our phylogenetic analysis suggests that it is a primordial eukaryotic invention that was retained in diverse groups, and independently lost several times in the evolution of other organisms. We demonstrate for the first time that the fungal Pet127 protein in vitro is a processive 5'-to-3' exoribonuclease capable of digesting various substrates in a sequence nonspecific manner. Mutations in conserved residues essential in the PD-(D/E)XK superfamily active site abolish the activity of Pet127. Deletion of the PET127 gene in the pathogenic yeast Candida albicans results in a moderate increase in the steady state levels of several transcripts and in accumulation of unspliced precursors and intronic sequences of three introns. Mutations in the active site residues result in a phenotype identical to that of the deletant, confirming that the exoribonuclease activity is related to the physiological role of the Pet127 protein. Pet127 activity is, however, not essential for maintaining the mitochondrial respiratory activity in C. albicans.

Keywords:  Candida albicans; Pet127; RNA degradation; exoribonuclease; mitochondria

DOI:  https://doi.org/10.1261/rna.079083.121
J Clin Invest. 2022 Feb 24. pii: e148852. [Epub ahead of print]

Brown adipose TRX2 deficiency activates mtDNA-NLRP3 to impair thermogenesis and protect against diet-induced insulin resistance.

Yanrui Huang, Jenny H Zhou, Haifeng Zhang, Alberto Canfrán-Duque, Abhishek K Singh, Rachel J Perry, Gerald Shulman, Carlos Fernandez-Hernando, Wang Min.

  Brown adipose tissue (BAT), a crucial heat-generating organ, regulate whole-body energy metabolism by mediating thermogenesis. BAT inflammation is implicated in the pathogenesis of mitochondrial dysfunction and impaired thermogenesis. However, the link between BAT inflammation and systematic metabolism remains unclear. Herein, we use mice with BAT deficiency of thioredoxin-2 (TRX2), a protein that scavenges mitochondrial reactive oxygen species (ROS), to evaluate the impact of BAT inflammation on metabolism and thermogenesis and its underlying mechanism. Our results describe that BAT-specific TRX2 ablation improves systematic metabolic performance via enhancing lipid uptake, which protects mice from diet-induced obesity, hypertriglyceridemia, and insulin resistance. TRX2 deficiency impairs adaptive thermogenesis by suppressing fatty acid oxidation. Mechanistically, loss of TRX2 induces excessive mitochondrial ROS, mitochondrial integrity disruption, and cytosolic release of mitochondrial DNA, which in turn activate aberrant innate immune responses in BAT, including the cGAS-STING and the NLRP3 inflammasome pathways. We identify NLRP3 as a key converging point, as its inhibition reverses both the thermogenesis defect and the metabolic benefits seen under nutrient overload in BAT-specific Trx2-deficient mice. In conclusion, we identify TRX2 as a critical hub integrating oxidative stress, inflammation, and lipid metabolism in BAT; uncovering an adaptive mechanism underlying the link between BAT inflammation and systematic metabolism.

Keywords:  Adipose tissue; Inflammation; Innate immunity; Metabolism; Mitochondria

DOI:  https://doi.org/10.1172/JCI148852
iScience. 2022 Feb 18. 25(2): 103823

Aberrant cyclin C nuclear release induces mitochondrial fragmentation and dysfunction in MED13L syndrome fibroblasts.

Kai-Ti Chang, Jan Jezek, Alicia N Campbell, David C Stieg, Zachary A Kiss, Kevin Kemper, Ping Jiang, Hyung-Ok Lee, Warren D Kruger, Peter M van Hasselt, Randy Strich.

  MED13L syndrome is a haploinsufficiency developmental disorder characterized by intellectual disability, heart malformation, and hypotonia. MED13L controls transcription by tethering the cyclin C-Cdk8 kinase module (CKM) to the Mediator complex. In addition, cyclin C has CKM-independent roles in the cytoplasm directing stress-induced mitochondrial fragmentation and regulated cell death. Unstressed MED13L S1497 F/fs patient fibroblasts exhibited aberrant cytoplasmic cyclin C localization, mitochondrial fragmentation, and a 6-fold reduction in respiration. In addition, the fibroblasts exhibited reduced mtDNA copy number, reduction in mitochondrial membrane integrity, and hypersensitivity to oxidative stress. Finally, transcriptional analysis of MED13L mutant fibroblasts revealed reduced mRNA levels for several genes necessary for normal mitochondrial function. Pharmacological or genetic approaches preventing cyclin C-mitochondrial localization corrected the fragmented mitochondrial phenotype and partially restored organelle function. In conclusion, this study found that mitochondrial dysfunction is an underlying defect in cells harboring the MED13L S1497 F/fs allele and identified cyclin C mis-localization as the likely cause. These results provide a new avenue for understanding this disorder.

Keywords:  Biochemistry; Biological sciences; Cell biology

DOI:  https://doi.org/10.1016/j.isci.2022.103823
Int J Mol Sci. 2022 Feb 18. pii: 2252. [Epub ahead of print]23(4):

Analysis of Mitochondrial Function, Structure, and Intracellular Organization In Situ in Cardiomyocytes and Skeletal Muscles.

Andrey V Kuznetsov, Sabzali Javadov, Raimund Margreiter, Judith Hagenbuchner, Michael J Ausserlechner.

  Analysis of the function, structure, and intracellular organization of mitochondria is important for elucidating energy metabolism and intracellular energy transfer. In addition, basic and clinically oriented studies that investigate organ/tissue/cell dysfunction in various human diseases, including myopathies, cardiac/brain ischemia-reperfusion injuries, neurodegenerative diseases, cancer, and aging, require precise estimation of mitochondrial function. It should be noted that the main metabolic and functional characteristics of mitochondria obtained in situ (in permeabilized cells and tissue samples) and in vitro (in isolated organelles) are quite different, thereby compromising interpretations of experimental and clinical data. These differences are explained by the existence of the mitochondrial network, which possesses multiple interactions between the cytoplasm and other subcellular organelles. Metabolic and functional crosstalk between mitochondria and extra-mitochondrial cellular environments plays a crucial role in the regulation of mitochondrial metabolism and physiology. Therefore, it is important to analyze mitochondria in vivo or in situ without their isolation from the natural cellular environment. This review summarizes previous studies and discusses existing approaches and methods for the analysis of mitochondrial function, structure, and intracellular organization in situ.

Keywords:  cardiac mitochondria; confocal fluorescent imaging; heterogeneity; mitochondrial intracellular organization; mitochondrial respiratory function; mitochondrial swelling; oxidative phosphorylation; reactive oxygen species

DOI:  https://doi.org/10.3390/ijms23042252
Biomolecules. 2022 Jan 19. pii: 162. [Epub ahead of print]12(2):

Bcl-2 Family Members and the Mitochondrial Import Machineries: The Roads to Death.

Lisenn Lalier, François Vallette, Stéphen Manon.

  The localization of Bcl-2 family members at the mitochondrial outer membrane (MOM) is a crucial step in the implementation of apoptosis. We review evidence showing the role of the components of the mitochondrial import machineries (translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM)) in the mitochondrial localization of Bcl-2 family members and how these machineries regulate the function of pro- and anti-apoptotic proteins in resting cells and in cells committed into apoptosis.

Keywords:  apoptosis; bcl-2 family; mitochondrial import machineries

DOI:  https://doi.org/10.3390/biom12020162
Free Radic Biol Med. 2022 Feb 17. pii: S0891-5849(22)00059-4. [Epub ahead of print]182 23-33

C19orf12 ablation causes ferroptosis in mitochondrial membrane protein-associated with neurodegeneration.

Changjuan Shao, Julia Zhu, Xiaopin Ma, Sandra L Siedlak, Mark L Cohen, Alan Lerner, Wenzhang Wang.

  Mitochondrial membrane protein-associated with neurodegeneration (MPAN) is a rare genetic disease characterized by aggressive neurodegeneration and massive iron accumulation in patients' brains. Genetics studies identified defects in C19orf12 locus being associated with MPAN which likely caused loss of function although underlying pathogenic mechanism(s) remain elusive. In the present study, we investigated C19orf12 knockout (KO) M17 neuronal cells and primary skin fibroblasts from MPAN patients with C19orf12 homozygous G58S or heterozygous C19orf12 p99fs*102 mutations as cellular models of MPAN. C19orf12 KO cells and MPAN fibroblast cells demonstrated mitochondrial fragmentation and dysfunction, iron overload and increased oxidative damage. Antioxidant NAC and iron chelator DFO rescued both oxidative stress and mitochondrial deficits. Moreover, C19orf12 KO cells and MPAN fibroblast cells were susceptible to erastin- or RSL3-induced ferroptosis which could be almost completely prevented by pretreatment of iron chelator DFO. Importantly, we also found mitochondrial fragmentation and increased ferroptosis related oxidative damage in neurons in the biopsied cortical tissues from an MPAN patient. Collectively, these results supported the notion that iron overload and ferroptosis likely play an important role in the pathogenesis of MPAN.

Keywords:  C19orf12; Ferroptosis; Iron accumulation; MPAN; Mitochondrial dysfunction; Oxidative stress

DOI:  https://doi.org/10.1016/j.freeradbiomed.2022.02.006
Redox Biol. 2022 Feb 06. pii: S2213-2317(22)00030-1. [Epub ahead of print]51 102258

Redox-dependent loss of flavin by mitochondria complex I is different in brain and heart.

Belem Yoval-Sánchez, Fariha Ansari, Joel James, Zoya Niatsetskaya, Sergey Sosunov, Peter Filipenko, Irina G Tikhonova, Vadim Ten, Ilka Wittig, Ruslan Rafikov, Alexander Galkin.

  Pathologies associated with tissue ischemia/reperfusion (I/R) in highly metabolizing organs such as the brain and heart are leading causes of death and disability in humans. Molecular mechanisms underlying mitochondrial dysfunction during acute injury in I/R are tissue-specific, but their details are not completely understood. A metabolic shift and accumulation of substrates of reverse electron transfer (RET) such as succinate are observed in tissue ischemia, making mitochondrial complex I of the respiratory chain (NADH:ubiquinone oxidoreductase) the most vulnerable enzyme to the following reperfusion. It has been shown that brain complex I is predisposed to losing its flavin mononucleotide (FMN) cofactor when maintained in the reduced state in conditions of RET both in vitro and in vivo. Here we investigated the process of redox-dependent dissociation of FMN from mitochondrial complex I in brain and heart mitochondria. In contrast to the brain enzyme, cardiac complex I does not lose FMN when reduced in RET conditions. We proposed that the different kinetics of FMN loss during RET is due to the presence of brain-specific long 50 kDa isoform of the NDUFV3 subunit of complex I, which is absent in the heart where only the canonical 10 kDa short isoform is found. Our simulation studies suggest that the long NDUFV3 isoform can reach toward the FMN binding pocket and affect the nucleotide affinity to the apoenzyme. For the first time, we demonstrated a potential functional role of tissue-specific isoforms of complex I, providing the distinct molecular mechanism of I/R-induced mitochondrial impairment in cardiac and cerebral tissues. By combining functional studies of intact complex I and molecular structure simulations, we defined the critical difference between the brain and heart enzyme and suggested insights into the redox-dependent inactivation mechanisms of complex I during I/R injury in both tissues.

Keywords:  Brain; Cardiac infarction; Flavin mononucleotide; Heart; Isoforms; Mitochondrial complex I; Reverse electron transfer; Stroke; Tissue-specificity

DOI:  https://doi.org/10.1016/j.redox.2022.102258
FEBS J. 2022 Feb 25.

Mitochondrial and metabolic remodeling in human skin fibroblasts in response to glucose availability.

Sónia A Pinho, Cláudio F Costa, Cláudia M Deus, Sonia L C Pinho, Inês Miranda-Santos, Gonçalo Afonso, Olivia Bagshaw, Jeffrey Stuart, Paulo J Oliveira, Teresa Cunha-Oliveira.

  Cell culture conditions highly influence cell metabolism in vitro. This is relevant for preclinical assays, for which fibroblasts are an interesting cell model, with applications in regenerative medicine, diagnostics and therapeutic development for personalized medicine, and the validation of ingredients for cosmetics. Given these cells' short lifespan in culture, we aimed to identify the best cell culture conditions and promising markers to study mitochondrial health and stress in Normal Human Dermal Fibroblasts (NHDF). We tested the effect of reducing glucose concentration in the cell medium from high glucose (HGm) to a more physiological level (LGm), or its complete removal and replacement by galactose (OXPHOSm), always in the presence of glutamine and pyruvate. We have demonstrated that only with OXPHOSm was it possible to observe the selective inhibition of mitochondrial ATP production. This reliance on mitochondrial ATP was accompanied by changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), oxidation of citric acid cycle substrates, fatty acids, lactate, and other substrates, increased mitochondrial network extension and polarization, increased protein content of VDAC and PGC1α and changes in several key transcripts related to energy metabolism. LGm did not promote significant metabolic changes in NHDF, although mitochondrial network extension and VDAC protein content were increased compared to HGm-cultured cells. Our results indicate that short-term adaptation to OXPHOSm is ideal for studying mitochondrial health and stress in NHDF.

Keywords:  bioenergetics; human fibroblasts; metabolic remodeling; metabolism; mitochondrial health

DOI:  https://doi.org/10.1111/febs.16413
Mov Disord. 2022 Feb 22.

Disruption of Mitochondrial Complex I Induces Progressive Parkinsonism.

Mario Cornejo-Olivas, Lesley Wu, Alastair Noyce.

DOI: https://doi.org/10.1002/mds.28961
J Cell Biol. 2022 04 04. pii: e202112104. [Epub ahead of print]221(4):

Stress-responsive regulation of extracellular proteostasis.

Jaleh S Mesgarzadeh, Joel N Buxbaum, R Luke Wiseman.

Genetic, environmental, and aging-related insults can promote the misfolding and subsequent aggregation of secreted proteins implicated in the pathogenesis of numerous diseases. This has led to considerable interest in understanding the molecular mechanisms responsible for regulating proteostasis in extracellular environments such as the blood and cerebrospinal fluid (CSF). Extracellular proteostasis is largely dictated by biological pathways comprising chaperones, folding enzymes, and degradation factors localized to the ER and extracellular space. These pathways limit the accumulation of nonnative, potentially aggregation-prone proteins in extracellular environments. Many reviews discuss the molecular mechanisms by which these pathways impact the conformational integrity of the secreted proteome. Here, we instead focus on describing the stress-responsive mechanisms responsible for adapting ER and extracellular proteostasis pathways to protect the secreted proteome from pathologic insults that challenge these environments. Further, we highlight new strategies to identify stress-responsive pathways involved in regulating extracellular proteostasis and describe the pathologic and therapeutic implications for these pathways in human disease.

DOI: https://doi.org/10.1083/jcb.202112104
Cell Mol Life Sci. 2022 Feb 26. 79(3): 155

ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation.

Hyun-Jung Kim, Woo-Jin Kim, Hye-Rim Shin, Hee-In Yoon, Jae-I Moon, Eunji Lee, Jin-Muk Lim, Young-Dan Cho, Mi-Hye Lee, Hong-Gee Kim, Hyun-Mo Ryoo.

  Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.

Keywords:  NFκB; Osteoblast; Peptidyl arginine deiminase 2; Reactive oxygen species; Senescence; Senescence-associated secretory phenotype

DOI:  https://doi.org/10.1007/s00018-022-04186-5
Genome Res. 2022 Feb 22.

Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation.

Qianru Li, Haiwang Yang, Emily K Stroup, Hongbin Wang, Zhe Ji.

We describe a low-input RNase footprinting approach for the rapid quantification of ribosome-protected fragments with as few as 1000 cultured cells. The assay uses a simplified procedure to selectively capture ribosome footprints based on optimized RNase digestion. It simultaneously maps cytosolic and mitochondrial translation with single-nucleotide resolution. We applied it to reveal selective functions of the elongation factor TUFM in mitochondrial translation, as well as synchronized repression of cytosolic translation after TUFM perturbation. We show the assay is applicable to small amounts of primary tissue samples with low protein synthesis rates, including snap-frozen tissues and immune cells from an individual's blood draw. We showed its feasibility to characterize the personalized immuno-translatome. Our analyses revealed that thousands of genes show lower translation efficiency in monocytes compared with lymphocytes, and identified thousands of translated noncanonical open reading frames (ORFs). Altogether, our RNase footprinting approach opens an avenue to assay transcriptome-wide translation using low-input samples from a wide range of physiological conditions.

DOI: https://doi.org/10.1101/gr.276139.121
Proc Natl Acad Sci U S A. 2022 Mar 01. pii: e2110357119. [Epub ahead of print]119(9):

Mitochondrial COA7 is a heme-binding protein with disulfide reductase activity, which acts in the early stages of complex IV assembly.

Luke E Formosa, Shadi Maghool, Alice J Sharpe, Boris Reljic, Linden Muellner-Wong, David A Stroud, Michael T Ryan, Megan J Maher.

  Cytochrome c oxidase (COX) assembly factor 7 (COA7) is a metazoan-specific assembly factor, critical for the biogenesis of mitochondrial complex IV (cytochrome c oxidase). Although mutations in COA7 have been linked to complex IV assembly defects and neurological conditions such as peripheral neuropathy, ataxia, and leukoencephalopathy, the precise role COA7 plays in the biogenesis of complex IV is not known. Here, we show that loss of COA7 blocks complex IV assembly after the initial step where the COX1 module is built, progression from which requires the incorporation of copper and addition of the COX2 and COX3 modules. The crystal structure of COA7, determined to 2.4 Å resolution, reveals a banana-shaped molecule composed of five helix-turn-helix (α/α) repeats, tethered by disulfide bonds. COA7 interacts transiently with the copper metallochaperones SCO1 and SCO2 and catalyzes the reduction of disulfide bonds within these proteins, which are crucial for copper relay to COX2. COA7 binds heme with micromolar affinity, through axial ligation to the central iron atom by histidine and methionine residues. We therefore propose that COA7 is a heme-binding disulfide reductase for regenerating the copper relay system that underpins complex IV assembly.

Keywords:  COA7; X-ray crystallography; cytochrome c oxidase; heme; mitochondria

DOI:  https://doi.org/10.1073/pnas.2110357119
Sci Rep. 2022 02 23. 12(1): 3045

Mitochondrial dysfunction associated with TANGO2 deficiency.

Paige Heiman, Al-Walid Mohsen, Anuradha Karunanidhi, Claudette St Croix, Simon Watkins, Erik Koppes, Richard Haas, Jerry Vockley, Lina Ghaloul-Gonzalez.

Transport and Golgi Organization protein 2 Homolog (TANGO2)-related disease is an autosomal recessive disorder caused by mutations in the TANGO2 gene. Symptoms typically manifest in early childhood and include developmental delay, stress-induced episodic rhabdomyolysis, and cardiac arrhythmias, along with severe metabolic crises including hypoglycemia, lactic acidosis, and hyperammonemia. Severity varies among and within families. Previous studies have reported contradictory evidence of mitochondrial dysfunction. Since the clinical symptoms and metabolic abnormalities are suggestive of a broad dysfunction of mitochondrial energy metabolism, we undertook a broad examination of mitochondrial bioenergetics in TANGO2 deficient patients utilizing skin fibroblasts derived from three patients exhibiting TANGO2-related disease. Functional studies revealed that TANGO2 protein was present in mitochondrial extracts of control cells but not patient cells. Superoxide production was increased in patient cells, while oxygen consumption rate, particularly under stress, along with relative ATP levels and β-oxidation of oleate were reduced. Our findings suggest that mitochondrial function should be assessed and monitored in all patients with TANGO2 mutation as targeted treatment of the energy dysfunction could improve outcome in this condition.

DOI: https://doi.org/10.1038/s41598-022-07076-9